When normal bladder cells are grown in culture they remain normal, and when diseased or abnormal bladder cells are grown in culture they remain abnormal. A patient needing bladder augmentation or regeneration likely does not have healthy bladder cells for regeneration, and therefore needs an alternative cell source. Because Bone Marrow Stromal Cells (BMSC) have the capacity to be cultured and differentiated into smooth muscle-like cells, they may be useful as an alternative cell source for tissue engineered bladder regeneration. BMSC are obtained by bone marrow aspiration and can be expanded to sufficient numbers in vitro. They are not stem cells, but display multiple features of a stem cell population, including similar cell proliferation, histological appearance, and contractile phenotype compared to primary smooth muscle cell (SMC) cultures. This application focuses on researching the promising potential of BMSC for bladder regeneration. First Specific Aim: (1) Characterize BMSC when co-cultured with bladder urothelial cells (UC) in vitro. (1.1) Isolate and culture BMSC, UC, and SMC, (1.2) Label BMSC with enhanced Green Fluorescent Protein (GFP) for later identification, (1.3) Co-culture BMSC with UC on SIS in vitro. (1.3.1) Assess effect of UC on BMSC cell growth and density, (1.3.2) Evaluate optimal timing to induce BMSC differentiation in UC co-culture on SIS through phenotypic appearance, molecular analysis, and immunohistochemical staining. Second Specific Aim: (2) Determine the in vivo survival of BMSC and their impact on bladder function and regeneration. (2.1) Label BMSC with enhanced GFP for later identification, (2.2) Co-culture GFP labeled BMSC and UC on SIS in vitro, (2.3) Implant cell-scaffold construct into a canine bladder after hemicystectomy, (2.3.1) Analyze bladder function and capacity using urodynamic studies, (2.3.2) Evaluate in vivo BMSC survival at different time points, (2.3.3) Evaluate extent of bladder regeneration at different time points through histology, contractility assays, and immunohistochemical staining. [unreadable] [unreadable] [unreadable]